P29 HEPATIC LIPID ACCUMULATION IN EXPERIMENTAL HEPARG MODEL FOR STEATOSIS INFLUENCES BUPROPION BIOTRANSFORMATION

Anitha Saravanakumar , University of Rhode Island, Kingston, RI
Enoch Cobbina , University of Rhode Island, Kingston, RI
Xin Bush , University of Rhode Island, Kingston, RI
Fatemeh Akhlaghi , Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI
Hepatic lipid accumulation (steatosis) is an early stage indicator that leads to spectrum of diseases such as non-alcoholic steatohepatitis (NASH), cirrhosis and fibrosis. Drug metabolism is impaired with lipid infiltration partly due to dysregulation in microsomal cytochrome (CYP) P450 activity1 . The present study tested the effect of lipid loading on CYP gene expression and activity towards the hydrophobic drug Bupropion (BUP). HepaRG cells, differentiated in-house were exposed to a mixture of saturated and unsaturated fatty acids, 0.5 mM palmitate and oleate (1:2) conjugated to BSA (0.02%) for 72h. Following the incubation, the model system was characterized using Oil Red O (ORO) staining for neutral lipids, tested for toxicity with WST-1 assay and triglyceride (TG) accumulation was measured using TG calorimetric assay. Increased lipid accumulation was noticed in ORO stain and the levels were significantly higher in treatment groups (P < 0.05, n=4). At 0.5 mM FA treatment, there was 25 times increase in triglycerides levels compared to as control similar to reported in-vivo data with no detectable toxicity (n=4)2. The detection of the Total AKT/Phospho AKT enabled us to confirm the reduced insulin signaling in the fatty-acid-loaded HepaRG cells as observed in the steatotic-human hepatocytes3. CYP2B6 enzyme activity was measured using BUP, in BSA-control as well as treatment HepaRG cells. The kinetic parameters of BUP were analyzed for BSA control (Km = 266.92; Vmax = 2.94 pmol/million/min; n=4) and FA-treatment (Km = 1260.10 ; Vmax = 1.99 nmol/million/min; n=4); in FA treatment the Km had increased by 6-folds and Vmaxwas reduced by 2-fold to that control. The results indicate both enzyme affinity and rate of enzyme activity was significantly changed in steatosis. Also, the gene expression levels were measured using Taqman probes. There was significant down-regulation in gene expression of PXR, CAR, CYP3A4, CYP2B6 but up-regulation in CYP2E1 (P < 0.05, n=4). Thus, we demonstrated that the accumulation of saturated and polyunsaturated fatty acids may selectively inhibit CYP2B6 pathway. In conjunction with inhibition, CYP-down regulation may contribute to impaired drug clearance during early liver injury.

[1] Kopec KL, Burns D. Nonalcoholic fatty liver disease: a review of the spectrum of disease, diagnosis, and therapy. Nutr Clin Pract 2011; 26: 565–76.

[2] Sunny NE, Parks EJ, Browning JD, Burgess SC. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab. 2011; 14:804–10.

[3] Cortez-Pinto H, de Moura M, Day C. Non-alcoholic steatohepatitis: from cell biology to clinical practice. J Hepatol 2006; 44: 197–208.